plasmid degradation in tris EDTA - (Sep/14/2007 )

I’m thoroughly mystified as to how around 1 in 4 or so of my positive cDNA plasmid clones seem to degrade as they sit in tris EDTA buffer after miniprep. The miniprep procedure I use is: resuspend in glucose/tris/EDTA with RNAseA; lyse with alkaline lysis buffer; neutralize with high salt (KOAc) solution-.( I don’t use phenol at the request of our core sequencing facility.) I then precipitate with ethanol and resuspend in tris EDTA.
I use E-coli JM109, which is endA-.
Other thing I have considered:
Residual lysis buffer perhaps from pipetter, but the pH of degrading prep seems to be fine.
Contaminated ethanol, but it seems all samples would affected, not just a few.
Could there be other bugs-maybe endA+-that take over when the amp breaks down in the overnight cultures? I grow them 12-16 hrs.

I’ve attached a pic of an agarose gel with 4 samples of RE digested clones. These were all prepped at the same time, yet 2 are degrading. Interestingly, if I run these samples without RE and buffer, they appear as a smear as well. How could DNAses function in TE?

well it seems your tris edta solution is out of date, i'm afraid of it.
it may affect samples differently, and that may came from sample concentration for exmample.
I would advice you to resuspend your samples in miliQ water for few tries and see if it's the point

and just on the off chance, perhaps you might want to reautoclave your plasmid ware.. ependorfs and pipette tips.
This is truely strange.

If you are preparing your own RNase A, it could have contaminating DNase activity.

Heat 10 mg/mL RNase A at 100°C. for 10 minutes to denature DNase I. Incubate 10 µL RNase A with 1 µg pBR322 cut with Msp I or Hinf I at 37°C. overnight. Check for residual DNase activity via PAGE. If residual DNase I activity exists, repeat the boiling step on more time. Recheck for contaminating DNase I. If residual activity remains, discard the prep and start over, as RNase A activity is inhibited if the process is repeated a 3rd time. Aliquot and store at –20°C.

Usually Mini preps with out phenol extraction step are proned to have DNase contamination.Therefore they tend to degrade soon.Therefore phenol extraction is recommended always.As you already mentioned that your core sequencin facility demands no phenol treatment the in such case y not go for kit prepatation of DNA atleast for the sequncing samples.
As for ur Tris EDTA sol, It seems to be fine coz not all samples show smear,only two have the smear .this is b'coz of diff amt of DNase contamination in diff samples during isolation.

From just a quick look at that gel, it looks like those 'degraded' samples are from minipreps that have been overloaded, allowing genomic DNA to come through. Try running some of the miniprep through another column and if that gets rid of the smear then problem solvered. Just add less lysate to your columns - they preferentially bind plasmid DNA, but if overloaded will capture genomic too.

Hope that is all it is.

Cheers,
Anil.

Hi,

When I run agrose gel of a miniprep plasmid, it usually shows two bands. I still remember I learnt this in class that supercoiled plasmids run differently from relaxed plasmids. And nicked plasmids also run differently. So I don't think this is degradation or genomic DNA...

To make sure it is degrading: put part of it at room temp of 37°C for longer time and run a gel of it. From the picture I can't tell if it is really degrading, maybe you just overloaded your gel? What's the concentration of your DNA for your different preps and how much did you put on gel? Might be genomic DNA as suggested as well.

Hi, I also think it might be different plasmid conformations but I can't find a reason for the smearing. Anyway, there's a group in my institution which works on plasmid purification and they keep telling everyone that plasmids are not as stable as one might think, depending even on their sequence!

I usually store my plasmids in nuclease-free water at -20ºC and have no problems with that.